The present invention relates to a protein capable of inducing a cytotoxic T cell (Cytotoxic T Lymphocytes, see xe2x80x9cIka Men""ekigaku (Medical Immunology), Revised 3rd Edition, Ed. by K. Kikuchi, also abbreviated as xe2x80x9cCTLxe2x80x9d hereinafter) against human gastric cancer cells in vivo or in vitro, and a DNA encoding the protein. Particularly, the present invention relates to a protein capable of presenting CTL against human gastric cancer cells by being bound to HLA-A31 antigen (Human Leucocyte Antigen, see xe2x80x9cGendai Men""ekigaku (Current Immunology)xe2x80x9d, 2nd Edition, Ed. By Y. Yamamura and T. Tada), and a DNA encoding the protein.
The present invention also relates to an agent for prevention or treatment of human gastric cancer, which comprises a protein capable of inducing CTL against human gastric cancer cells in vivo or in vitro, and a vaccine for prevention or treatment of human gastric cancer, which comprises a recombinant virus or a recombinant bacterium containing a DNA encoding the protein.
As the therapies for malignancy, in addition to surgical treatment, radiotherapy, and chemotherapy, there has been attempted immunotherapy which aims at obtaining therapeutic effect by enhancing the immune function of host patients. However, most of the immunotherapy procedures practically used thus far have obtained the effect by non-specifically enhancing immunecompetence of host patients, and drugs capable of inducing complete cure of tumors in clinical cases have not been practically used yet.
Many researchers have conducted investigations utilizing animal tumor models mainly based on mice to develop a drug capable of completely curing tumors. As a result, it has been clarified that tumors may be completely cured by efficiently inducing antigen specific immune responses, in particular, inducing cytotoxic T cells (CTLs), against tumor-associated antigens or tumor specific antigens expressed on various tumor cells. In order to treat tumors by such CTL induction, it is essential to elucidate an amino acid sequence of protein recognized by tumor specific CTL and a DNA sequence encoding the protein for each tumor.
In recent years, also for clinical tumors, it has been attempted to identify an antigen inducing tumor specific CTL and a DNA encoding the antigen, and utilize them for therapy.
Based on such conception, tumor antigen proteins capable of inducing CTL and DNAs encoding such proteins have been searched for. However, those identified so far are limited to the proteins such as those of MAGE family (T. Boon et al., Immunology Today, 18, 267-278, 1997), Mart-1, Thyrosinase, gp100 (S. A. Rosenberg, Immunology Today, 18, 175-182), which are the tumor antigens present in melanoma, and DNAs encoding them, and the proteins present in epidermoid cancer (derived from human head and neck cancer, CASP-8, J. Exp. Med., 186, 785-793, 1997) and DNAs encoding them.
On the other hand, as for digestive tract cancers including gastric cancer, the presence of a tumor antigen peptide which can induce CTL has been identified by the present inventors (Japanese Patent Unexamined Publication [KOKAI] No. Hei 9-151200/1997). However, it has not been clarified at all what kind of protein from which tumor peptide is derived, and much less structure of DNA encoding the tumor antigen protein.
Accordingly, the object of the present invention is to provide 1) a DNA sequence encoding a protein which can induce immune response against human gastric cancer, 2) a protein sequence encoded by the aforementioned DNA, 3) a vaccine for treatment or prevention of human gastric cancer which comprises a recombinant virus or a recombinant bacterium containing the aforementioned DNA, and 4) an agent for treatment or prevention of human gastric cancer which comprises the aforementioned protein.
The present inventors noted that CTL recognizing tumor antigens played an important role as a biological defense mechanism against tumor cells. That is, the present inventors noted that efficient induction of CTL by utilizing a protein usable as a part of vaccine or a DNA encoding such a protein was effective for treatment and prevention of tumors, and conducted studies.
It has been known that the CTL induction against tumor antigens is achieved through specific expression of tumor antigen genes in tumor cells and presentation of antigenic peptides derived from the tumor antigen proteins on cell surfaces together with HLA antigens on tumor cells. Therefore, the present inventors attemped to identify a gene that is specifically expressed in a gastric cancer cell line capable of inducing CTL specific for gastric cancer cells.
As described above, the present inventors identified a tumor antigen peptide (also referred to as xe2x80x9cF4.2 peptidexe2x80x9d hereinafter) which can induce CTL against digestive tract cancers including gastric cancer, and elucidated its amino acid sequence (Japanese Patent Unexamined Publication [KOKAI] No. Hei 9-151200/1997). Therefore, they attempted to isolate a cDNA encoding a protein from which the peptide is derived from a cDNA library of gastric cancer cell line by hybridization technique utilizing that amino acid sequence. As a result, several hybridization positive cDNA clones were identified. From these clones, those that can be amplified by PCR utilizing primers corresponding to the sequence of F4.2 peptide were further selected. When nucleotide sequences of all provided clones were determined, however, any clone encoding a sequence containing an amino acid sequence exactly the same as F4.2 peptide was not found, while several clones containing partial sequences of F4.2 peptide were selected.
On the other hand, other than the hybridization technique mentioned above, the present inventors developed a method utilizing phenotypic alteration caused by gene transfer as an index as a method for identifying a tumor antigen gene capable of inducing CTL specific for a gastric cancer cell. That is, the present inventors conceived that a target tumor antigen gene could be identified by introducing a chromosome DNA fragment derived from a suitable cell line, or cDNA derived from gastric cancer cell line that can induce gastric cancer antigen specific CTL, into a cell line that cannot induce CTL so that the gene should be expressed in the cell, and selecting a transgenic clone that has acquired the ability to induce CTL.
The present inventors has already successfully established a CTL cell line (Tc-HST-2) derived from a gastric cancer patient, which specifically respond to tumor antigen restricted to HLA-A31, and a gastric cancer cell line (HST-2) derived from the same patient, which is recognized by the CTL cell line (J. Immunol. Meth., 154; 235-243, 1992, Cancer, 75; 1484-1489, 1995). The gastric cancer cell line expresses HLA-A31 antigen. They have also succeeded in establishing a cell line (HOBC8-A31 cell) that expresses HLA-A31 antigen, but is not recognized by Tc-HST2 cells, and can be cultured in vitro. It is not expected that a gastric cancer antigen gene that can be expressed by HST-2 is expressed in that HOBC8-A31 cell.
When the aforementioned method utilizing phenotypic alteration as an index was applied to a cDNA clone encoding an amino acid sequence which contained a part of F4.2 peptide by using the aforementioned cell lines, it was found that a protein encoded by the cDNA activated the CTL cell line Tc-HST-2. Further, it was found that the cDNA was very useful as an agent for treatment and prevention of gastric cancer.
The present invention has been completed based on these findings.
That is, the present invention provides:
(1) A DNA which encodes a gastric cancer antigen protein present in a human gastric cancer cell (also referred to as the xe2x80x9cDNA of the present inventionxe2x80x9d hereinafter).
(2) The DNA of above (1), which encodes a gastric cancer antigen protein which comprises an amino acid sequence of SEQ ID NO: 2.
(3) The DNA of above (1), which encodes a gastric cancer antigen protein which comprises an amino acid sequence of SEQ ID NO: 2 having substitution, deletion, insertion, addition or inversion of one or several amino acids, and which can activate a cytotoxic T cell recognizing a gastric cancer antigen protein.
(4) The DNA of above (2) which comprises at least nucleotides corresponding to the nucleotide numbers 46-534 of the nucleotide sequence of SEQ ID NO: 1.
(5) The DNA of above (2) which is hybridizable with a nucleotide sequence comprising at least nucleotides corresponding to the nucleotide numbers 46-534 of the nucleotide sequence of SEQ ID NO: 1 under a stringent condition, and which encodes a protein that can activate a cytotoxic T cell recognizing a gastric cancer antigen protein.
(6) The DNA of above (5), wherein the stringent condition is a condition in which washing is performed at 60xc2x0 C., and at a salt concentration corresponding to 1xc3x97SSC and 0.1% SDS.
(7) The DNA of above (1), wherein the gastric cancer antigen protein encoded by the DNA comprises a partially modified amino acid sequence for realizing more efficient induction of a cytotoxic T cell recognizing the protein.
(8) A DNA which encodes a peptide containing an amino acid sequence comprising at least amino acids corresponding to amino acid numbers 62-70 of SEQ ID NO: 2, and being able to activate a cytotoxic T cell recognizing a gastric cancer antigen protein.
(9) A gastric cancer antigen protein which comprises an amino acid sequence of SEQ ID NO: 2.
(10) A gastric cancer antigen protein which comprises an amino acid sequence of SEQ ID NO: 2 having substitution, deletion, insertion, addition or inversion of one or several amino acids, and which can activate a cytotoxic T cell recognizing a gastric cancer antigen protein.
(11) A peptide which contains an amino acid sequence comprising at least amino acids corresponding to amino acid numbers 62-70 of SEQ ID NO: 2, and which can activate a cytotoxic T cell recognizing a gastric cancer antigen protein.
(12) A vaccine for prevention or treatment of human gastric cancer, which comprises a recombinant virus or a recombinant bacterium containing a DNA of any one of claims 1-8.
(13) An agent for prevention or treatment of human gastric cancer, which comprises the protein of above (9) or (10), or the peptide of above (11).